Alzheimer's disease (AD) is a currently-incurable progressive neurodegenerative disease most widely believed to develop as a result of decreased clearance from the brain of amyloid-? (A?), which is toxic to neuron function. According to the Alzheimer's Association, approximately 5.4 million people in the U.S. are living with AD in 2012, and the rate of new cases is increasing. While there is considerable knowledge on AD pathology and biochemistry, no clear cause for the onset of sporadic AD cases (>95% of the total) or biomarkers to indicate early stages of AD have been identified. The proposed work explores the role of exosomes, small lipid vesicles produced by brain cells and enriched with the sphingolipid ceramide, in controlling the amount of A? in the brain. Published data demonstrate that astrocytes secrete exosomes, a process dependent upon neutral sphingomyelinase-2 (nSMase2, which converts sphingomyelin to ceramide) following exposure to A?. New data suggest that astrocyte-derived exosomes facilitate uptake of A? into astrocytes and microglia under normal conditions, but during AD, exosomes in excessive numbers with increased levels of A? may also promote deposition of A? plaques. The goal of the proposed work is to clarify the role of exosomes in the progression of AD. The following specific aims are proposed: 1) Test the hypothesis that astrocyte-derived exosomes promote uptake and degradation of A? by neurons, astrocytes and microglia and 2) Test the hypothesis that altering exosome generation will reduce A? levels and plaque formation in vivo. Fluorescently labeled exosomes will be tested for their ability to bind and promote uptake of A? into wildtype astrocytes and microglia. The role of exosome-associated apoE on the uptake of A? and the role of nSMase2 in the uptake of A?-associated exosomes by neurons, astrocytes and microglia will be determined using primary cells from apoE- and nSMase2-deficient mice. The role of exosomes in the progression of AD pathology in vivo will be tested twofold: a) treatment of a robust AD mouse model (5xFAD) with pharmacological agents to inhibit nSMase2 and other enzymes that control ceramide levels that can influence exosome secretion and b) genetic ablation of nSMase2 in the 5xFAD model background, using conventional (fro/fro) and conditional knockouts. A? levels from brain and blood will be monitored by ELISA and plaque load determined by histochemistry. Results from these experiments will define a novel mechanism by which ceramide-enriched, A?-associated exosomes promote amyloid clearance from normal brains, but may also promote plaque formation when exosome uptake is impaired. Therefore, this proposal will have significant impact on AD by providing fundamental insight into the role of exosomes in progression of the disease and provide potential new therapeutic targets.